title: Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct.
abstract: BACKGROUND
  Interleukin-10 homologues encoded by Herpes viruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) hold interesting structural and biological characteristics compared to human interleukin-10 (hIL-10) that render these proteins promising candidates for therapeutic application in inflammatory bowel disease (IBD). Intestinal delivery of cytokines using bacterial carriers as chassis represents a novel approach for treatment of IBD patients. For proof of concept, a Sec-dependent transporter construct was designed for secretory expression of recombinant viral IL-10 proteins in the periplasm of Escherichia coli laboratory strain BL21 (DE3), which might serve as part of a prospective lysis based delivery and containment system.
  
  
  RESULTS
  The signal peptide of E. coli outer membrane protein F fused to the mature form of the viral IL-10 proteins enabled successful transport into the periplasm, a compartment which seems crucial for proper assembly of the dimeric configuration of the cytokines. Cytokine concentrations in different bacterial compartments were determined by ELISA and achieved yields of 67.8 ng/ml ± 24.9 ng/ml for HCMV IL-10 and 1.5 μg/ml ± 841.4 ng/ml for EBV IL-10 in the periplasm. Immunoblot analysis was used to confirm the correct size of the E. coli-derived recombinant cytokines. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the signal transduction cascade after IL-10 receptor interaction, as well as suppression of tumor necrosis factor α (TNF-α) release of lipopolysaccharide-stimulated mouse macrophages were used as read-out assays for proving in vitro biological activity of the E. coli derived, recombinant viral IL-10 counterparts.
  
  
  CONCLUSIONS
  In this study, proof of principle is provided that E. coli cells are a suitable chassis for secretory expression of viral IL-10 cytokines encoded by codon-optimized synthetic genes fused to the E. coli ompF signal sequence. In vitro biological activity evidenced by activation of transcription factor STAT3 and suppression of TNF-α in mammalian cell lines was shown to be strictly dependent on export of viral IL-10 proteins into the periplasmic compartment. E. coli might serve as carrier system for in situ delivery of therapeutic molecules in the gut, thus representing a further step in the development of novel approaches for treatment of IBD.
authors.affiliation: Institute of Medical Microbiology and Hygiene, TU Dresden, Fiedlerstrasse 42, 01307 Dresden, Germany. florian.gunzer@tu-dresden.de.
authors.forename: Sarah
authors.initials: S
authors.lastname: Förster
authors.forename: Manuela
authors.initials: M
authors.lastname: Brandt
authors.forename: Dorothea S
authors.initials: DS
authors.lastname: Mottok
authors.forename: Anke
authors.initials: A
authors.lastname: Zschüttig
authors.forename: Kurt
authors.initials: K
authors.lastname: Zimmermann
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Blattner
authors.forename: Florian
authors.initials: F
authors.lastname: Gunzer
authors.forename: Christoph
authors.initials: C
authors.lastname: Pöhlmann
mesh_terms: D000818:Animals; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000628:therapy; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019897:Periplasm / Q000378:metabolism; D010766:Phosphorylation; D018272:Porins / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C109237:OmpF protein; D018272:Porins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; D016753:Interleukin-10